Transglutaminases are a family of enzymes that catalyze the calcium-dependent, post-translational modification of the .gamma.-carboxamide group of peptide-bound glutamine residues. A key intermediate in the catalysis is a thioester acyl-enzyme complex. An .epsilon.-amino group of peptide-bound lysine is the acyl acceptor in protein crosslinking reactions: ##STR2## Alternatively, a free amine, such as putrescine, may act as an acyl acceptor resulting in the post-translational modification of proteins.
Transglutaminases have been implicated in a variety of disease states, including acne, psoriasis, cataracts, immunologic disease states, Alzheimer's disease and hyaline membrane disease.
For example, regarding the acne state, changes in transglutaminase activity during comedogenesis have been demonstrated by DeYoung et al., in J. Investigative Dermatology, 82, 275 (1984). These investigators have demonstrated that in early acne lesions there is intense transglutaminase activity in the involved sebaceous follicles. In normal follicles no such activity is observed. Furthermore, Dalziel et al., in Br. J. Exp. Pathology 65, 107-115 (1984) have shown that the cornified cell envelope, a product of transglutaminase activity, produces chronic inflammation when intradermally injected. The cornified envelope is responsible for the rigid, resistant structure of differentiated squamous cells. The cornified envelopes in acne comedones play an important role in the resistant cohesive nature of these structures and in their inflammatory potential upon rupture. Therefore, a need exists for an inhibitor of transglutaminase effective in the suppression of cornified envelope formation.
With regard to psoriasis, Bernard et al. in British Journal of Dermatology, 114, 279 (1986) have demonstrated, by histochemical activity staining, the precocious distribution of transglutaminase activity down to the suprabasal layer of involved psoriatic epidermis. In addition, the distribution of involucrin, one of the major substrates for epidermal transglutaminase, matches the distribution of transglutaminase activity. Thus, in psoriasis, there is an apparent loss of integrated control of the independent pathways for terminal differentiation of keratinocytes, and the onset of involucrin and transglutaminase activity is favored. A need exists for effective transglutaminase inhibitors to modulate the elevated transglutaminase activity in psoriatic epidermis.
Hereditary cataractous rat lenses show significantly elevated transglutaminase activities (2.7 to 17.7 times higher specific activities for young and old animals respectively). See Azari, P. et al., Current Eye Res, 1, 463 (1981).
With respect to immunologic disease states, much research has been directed to the role of transglutaminases in receptor functions, mitogen-induced cell activation and immune recognition. See Fesus, L. Surv. Immunol. Res. 1, 297-304 (1982).
Regarding Alzheimer's disease, neuronal transglutaminase is a likely mediator of the apparently irreversible crosslinking of neuronal proteins leading to the formation of paired helical fragments, which are a distinctive pathological feature of Alzheimer's disease. See Selkoe, et al., Science, 215, 1243-1245 (1982), Selkoe, et al., Proc. Natl. Acad. Sci., U.S.A., 79, 6070-6074 (1982), and Schlaepfer, "Biological Aspects of Alzheimer's Disease," (Ed. Katzman) p. 155, Cold Spring Harbour Laboratory. Bundles of paired helical fragments form neural fibrillary tangles, and the number of neuritic plaques correlates to the degree of intellectual impairment. See Farmer, et al. in J. Neuropathol. Exp. Neurol. 35, 367 (1976).
Previously reported inhibitors of transglutaminase include alternate substrate inhibitors, covalent inactivators and active site directed inhibitors. The alternate substrate inhibitors include alkyl primary amines, such as monodansylcadaverine, and alternative acyl-donors, such as beta-phenyl propionylthiocholine. These inhibitors prevent protein crosslinking, but do not prevent post-translational modification of proteins. They suffer from the drawback that they are effective only in relatively high concentrations, i.e., at 10.sup.-3 M or higher. The covalent inhibitors include alkyl isocyanates, such as (CH.sub.3).sub.2 --CH--CH.sub.2 --N.dbd.C.dbd.O, as titrants of active site cysteine residues, but these lack specificity for transglutaminases. An active site directed inhibitor is cystamine, which lacks specificity for transglutaminases and is effective only at concentrations of greater than 10.sup.-3 M.
Accordingly, a need exists for specific and potent inactivators of transglutaminases.